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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)

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Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
  • Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
  • Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
  • Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
  • Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [EPR13028(B)] to OTUB1 - BSA and Azide free
  • Suitable for: WB, IP
  • Knockout validated
  • Reacts with: Mouse, Rat, Human

Conjugates logo Related conjugates and formulations

Unconjugated

Overview

  • Product name

    Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free
    See all OTUB1 primary antibodies

  • Description

    Rabbit monoclonal [EPR13028(B)] to OTUB1 - BSA and Azide free

  • Host species

    Rabbit

  • Tested applications

    Suitable for: WB, IPmore details
    Unsuitable for: Flow Cyt,ICC/IF or IHC-P

  • Species reactivity

    Reacts with: Mouse, Rat, Human

  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Wild-type HAP1, HeLa, MCF7, HepG2, HEK-293T, and HEK-293 cell lysates. Rat and mouse heart tissue lysates. IP: HeLa cell lysate.

  • General notes

    ab232581 is the carrier-free version of ab175200.

    Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.

    This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

    Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

    This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.

Properties

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab232581 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 31 kDa.
IP
Use at an assay dependent concentration.
Notes
WB
Use at an assay dependent concentration. Predicted molecular weight: 31 kDa.
IP
Use at an assay dependent concentration.
Application notes
Is unsuitable for Flow Cyt,ICC/IF or IHC-P.

Target

  • Function

    Hydrolase that can remove conjugated ubiquitin from proteins and plays an important regulatory role at the level of protein turnover by preventing degradation. Regulator of T-cell anergy, a phenomenon that occurs when T-cells are rendered unresponsive to antigen rechallenge and no longer respond to their cognate antigen. Acts via its interaction with RNF128/GRAIL, a crucial inductor of CD4 T-cell anergy. Isoform 1 destabilizes RNF128, leading to prevent anergy. In contrast, isoform 2 stabilizes RNF128 and promotes anergy. Surprisingly, it regulates RNF128-mediated ubiquitination, but does not deubiquitinate polyubiquitinated RNF128. Deubiquitinates estrogen receptor alpha (ESR1). Mediates deubiquitination of 'Lys-48'-linked polyubiquitin chains, but not 'Lys-63'-linked polyubiquitin chains. Not able to cleave di-ubiquitin. Also capable of removing NEDD8 from NEDD8 conjugates, but with a nuch lower preference compared to 'Lys-48'-linked ubiquitin.

  • Tissue specificity

    Isoform 1 is ubiquitous. Isoform 2 is expressed only in lymphoid tissues such as tonsils, lymph nodes and spleen, as well as peripheral blood mononuclear cells.

  • Sequence similarities

    Belongs to the peptidase C65 family.
    Contains 1 OTU domain.

  • Domain

    In addition to ubiquitin-binding at the Cys-91 active site, a proximal ubiquitin-binding site is also present at Cys-23 Occupancy of the active site is needed to enable tight binding to the second site. Distinct binding sites for the ubiquitins may allow to discriminate among different isopeptide linkages (i.e. 'Lys-48'-, 'Lys-63'-linked polyubiquitin) in polyubiquitin substrates and achieve linkage-specific deubiquitination.

  • Cellular localization

    Cytoplasm.
  • Information by UniProt

  • Database links

    see all

  • Alternative names

    • Deubiquitinating enzyme OTUB1 antibody
    • hOTU1 antibody
    • HSPC263 antibody
    • OTB1 antibody
    • OTU domain containing ubiquitin aldehyde binding protein 1 antibody
    • OTU domain, ubiquitin aldehyde binding 1 antibody
    • OTU domain-containing Ubal-binding protein 1 antibody
    • OTU domain-containing ubiquitin aldehyde-binding protein 1 antibody
    • OTU-domain Ubal-binding 1 antibody
    • OTU1 antibody
    • Otub1 antibody
    • OTUB1_HUMAN antibody
    • Otubain 1 antibody
    • Otubain-1 antibody
    • Ubiquitin specific processing protease OTUB1 antibody
    • Ubiquitin thioesterase OTUB1 antibody
    • ubiquitin-specific protease otubain 1 antibody
    • Ubiquitin-specific-processing protease OTUB1 antibody
    see all

Images

  • Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
    Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
    All lanes : Anti-OTUB1 antibody [EPR13028(B)] (ab175200) at 1/1000 dilution

    Lane 1 : Wild-type HEK-293T cell lysate
    Lane 2 : OTUB1 knockout HEK-293T cell lysate

    Lysates/proteins at 20 µg per lane.

    Performed under reducing conditions.

    Predicted band size: 31 kDa
    Observed band size: 130 kDa why is the actual band size different from the predicted?



    This data was developed using the same antibody clone in a different buffer formulation (ab175200).

    Lanes 1-2: Merged signal (red and green). Green - ab175200 observed at 31 kDa. Red - loading control ab8245 observed at 37 kDa.

     ab175200 Anti-OTUB1 antibody [EPR13028(B)] was shown to specifically react with OTUB1 in wild-type HEK-293T cells. Loss of signal was observed when knockout cell line ab266551 (knockout cell lysate ab257569) was used. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab175200 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°CC at 1 in 1000 Dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

     

     

  • Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
    Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)

    ab175200 (purified) at 1:50 dilution (2ug) immunoprecipitating OTUB1 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate.
    Lane 1 (input): HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10ug
    Lane 2 (+): ab175200 & HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
    Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab175200 in HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate
    For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
    Blocking and diluting buffer: 5% NFDM/TBST.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175200).

  • Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
    Immunoprecipitation - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)

    Western blot analysis on immunoprecipitation pellet from MCF7 cell lysate labeling OTUB1 with unpurified ab175200 at 1/10 dilution.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175200).

  • Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
    Western blot - Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
    All lanes : Anti-OTUB1 antibody [EPR13028(B)] (ab175200) at 1/1000 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : OTUB1 knockout HAP1 whole cell lysate
    Lane 3 : HeLa whole cell lysate
    Lane 4 : HEK-293 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 31 kDa



    Lanes 1 - 4: Merged signal (red and green). Green - ab175200 observed at 35 kDa. Red - loading control, ab18058, observed at 130 kDa.

    ab175200 was shown to specifically react with OTUB1 in wild type cells as signal was lost in OTUB1 knockout cells. Wild-type and OTUB1 knockout samples were subjected to SDS-PAGE. ab175200 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1,000 dilution and 1/20,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20,000 dilution for 1 hour at room temperature before imaging.

    This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab175200).

     

  • Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)
    Anti-OTUB1 antibody [EPR13028(B)] - BSA and Azide free (ab232581)

Protocols

To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

Click here to view the general protocols

References (0)

Publishing research using ab232581? Please let us know so that we can cite the reference in this datasheet.

ab232581 has not yet been referenced specifically in any publications.

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